The Tat (Twin-arginine translocatin) system is a recently defined protein export pathway that serves to translocate folded proteins. The substrates of the Tat pathway contain specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif-S/T-R-R-x-F-L-X. Here is the report of knocked out the tatA, tatB and tatC genes of the V. cholerae by suicide plasmid homologous recombination technology. Mutant strains showed obvious changes of growth characteristics. The transport of a typical Tat pathway substrate, trimethylamine N-oxide reductase (molybdoenzyme), was completely blocked. The physiochemical reactions of the parent and mutant strains were also analyzed. Four physiochemical reactions using D-galactose, L-asparagine, glyl-L-aspartic acid and D-L-alpha-glycerol phosphate as substrates were negative in mutant strains, which might be affected by the inactivation of the Tat-dependent system.